Novel tandem antibacterial peptide CC28 as well as recombinant engineering bacteria and application thereof

Abstract

The invention provides a novel tandem antibacterial peptide CC28 as well as a recombinant engineering bacterium and application thereof, and belongs to the technical field of bioengineering and animal nutrition and feed. The invention provides a novel tandem antibacterial peptide CC28. The amino acid sequence of the novel tandem antibacterial peptide CC28 is shown as SEQ ID NO. 1. The novel tandem antibacterial peptide CC28 has good bacteriostatic and bactericidal effects on gram-negative bacteria and gram-positive bacteria, has low hemolytic activity on rabbits and human beings, and has the effects of killing mouse breast cancer cells 4T1.2, human uterine cancer HeLa cells, human breast cancer cells MCF7 and MDA-MB-231. The antibacterial peptide CC28 provided by the invention and ligand cellulase are fused and inserted into a pichia expression vector pGAPZ alpha A, and are electrically transferred into SMD1168 to obtain a high-expression recombinant pichia pastoris engineering strain, and powder prepared by spray-drying fermentation liquor of the recombinant pichia pastoris engineering strain can be applied to poultry and has the effects of promoting growth, nutrition and immunity.

Description

Technical Field

[0001] The present invention relates to the fields of bioengineering, animal nutrition and feed technology, and in particular to a novel tandem antimicrobial peptide CC28, a recombinant engineered bacterium and applications thereof. Background Art

[0002] So far, it has been found that antimicrobial peptides (AMPs) (14 to 60 amino acid residues) have a wide range of functions. In terms of antimicrobial activity, they have no drug residues and are not prone to the development of resistant bacteria, making them good candidates for the development of new alternative antibiotics. AMPs also have limitations. Early developed antimicrobial peptides have problems with low activity, high cytotoxicity and hemolytic activity, and are susceptible to protease degradation. In addition, the high cost of synthesis makes it impossible to mass-produce antimicrobial peptides for livestock applications. Bioengineering methods are the most effective methods for producing protein, peptide or enzyme yields. Pichia pastoris is one of the most commonly used high-efficiency eukaryotic expression systems. However, in order to achieve effective antimicrobial peptide production, some challenges need to be overcome, such as avoiding the toxicity of antimicrobial peptides to host strains and expression quantification.

[0003] Therefore, in order to obtain higher quality and yield production, the present invention designs a highly active peptide, and obtains a high-expression strain by connecting and fusing it with a cellulase ligand. It has low-carbon and multifunctional benefits of antibacterial and increased nutritional activity, and has significant benefits for feed resource development. Summary of the Invention

[0004] The purpose of the present invention is to provide a highly active peptide with good antibacterial effect, and to obtain a high-expression strain by connecting and fusing it with a cellulase ligand, thereby reducing the difficulty of obtaining the highly active peptide.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a novel tandem antimicrobial peptide CC28, the amino acid sequence of which is shown in SEQ ID NO.1; and the nucleotide sequence of which is shown in SEQ ID NO.3.

[0007] The present invention provides a CC28-cellulase protein, which is assembled from an antimicrobial peptide CC28 and a ligand cellulase. The amino acid sequence of the CC28-cellulase protein is shown in SEQ ID NO.2, and the nucleotide sequence of the CC28-cellulase protein is shown in SEQ ID NO.6, which includes the antimicrobial peptide CC28 nucleotide sequence and the ligand sequence.

[0008] The present invention provides a recombinant Pichia pastoris engineered bacterium that secretes the CC28-cellulase protein. The preparation method comprises the following steps: subcloning the target protein gene into the pGAPZαA expression vector to construct a recombinant plasmid pGAPZαA / CC28-CE, and then transferring the recombinant plasmid into Pichia pastoris to obtain the recombinant Pichia pastoris engineered bacterium;

[0009] The target protein gene is shown in SEQ ID NO.6.

[0010] The invention provides a recombinant plasmid pGAPZαA / CC28-CE.

[0011] The present invention provides the use of the novel tandem antimicrobial peptide CC28 or the CC28-cellulase protein or the recombinant Pichia pastoris engineered bacteria or the recombinant plasmid pGAPZαA / CC28-CE in the preparation of antimicrobial products;

[0012] Preferably, the bacteria targeted by the antibacterial product include Gram-negative bacteria and / or Gram-positive bacteria.

[0013] Preferably, the Gram-negative bacteria include one or more of Escherichia coli, Salmonella enterica and Enterobacter aerogenes; and the Gram-positive bacteria include Streptococcus agalactiae and / or Staphylococcus aureus.

[0014] Preferably, the MIC value of the novel tandem antimicrobial peptide CC28 in the antimicrobial product against the Gram-negative bacteria and the Gram-positive bacteria is 1.11 μmol / L to 71.1 μmol / L.

[0015] The present invention also provides the use of the novel tandem antimicrobial peptide CC28 or the CC28-cellulase protein or the recombinant Pichia pastoris engineered bacteria or the recombinant plasmid pGAPZαA / CC28-CE in the preparation of livestock feed additives.

[0016] The present invention also provides the use of the novel tandem antimicrobial peptide CC28 or the CC28-cellulase protein or the recombinant Pichia pastoris engineered bacteria or the recombinant plasmid pGAPZαA / CC28-CE in the preparation of anticancer drugs.

[0017] The novel tandem antimicrobial peptide CC28 provided by the present invention has good antibacterial and bactericidal effects on both Gram-negative and Gram-positive bacteria, with minimum inhibitory concentrations for Escherichia coli and Clostridium perfringens reaching 1.1 and 2.2 μmol / L, respectively. It has low hemolytic activity against rabbits and humans, and has killing effects on mouse breast cancer cells 4T1.2, human uterine cancer HeLa cells, and human breast cancer cells MCF7 and MDA-MB-231. It can be used as an antibacterial agent or anticancer drug.

[0018] In addition, the yeast strain of the pGAPZαA system does not produce toxins when used to produce feed additives and is food safety level. The antimicrobial peptide expressed and secreted by Pichia pastoris contains a ligand protein cellulase structure that can prevent the hydrolysis of the antimicrobial peptide and weaken the toxicity of CC28, thereby promoting feed digestion. At the same time, the recombinant gene provided by the present invention improves the secretion of the exogenous protein of the antimicrobial peptide CC28 expressed by Lactococcus lactis, which will provide technical support for the large-scale production of feed additives and engineered lactic acid bacteria preparations.

[0019] The engineered bacteria preparation method provided by the present invention is simple, rapid, and efficient. The target protein is secreted and expressed in the culture medium, facilitating isolation and purification. The powder produced by spray drying is applied to poultry. It can improve poultry growth performance, nutrient utilization, slaughter performance, and fecal microbial flora. It can be used as a new feed additive in poultry production and has promising industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the predicted structure of the antimicrobial peptide CC283D;

[0021] Figure 2 is the peptide mass spectrum of the antimicrobial peptide CC28;

[0022] Figure 3 This is the result of the effect of antimicrobial peptide CC28 on mouse breast cancer cells 4T1.2;

[0023] Figure 4 This is the result of the effect of antimicrobial peptide CC28 on human uterine cancer cells Hela;

[0024] Figure 5 The effect of antimicrobial peptide CC28 on human breast cancer cells MCF-7;

[0025] Figure 6 The effect of antimicrobial peptide CC28 on human breast cancer cells MDA-MB-231;

[0026] Figure 7 The results of double enzyme digestion of the recombinant vector are shown in A, the results of PCR identification are shown in B, and the results of linearization are shown in C;

[0027] Figure 8 Screening Zeocin for the present invention TM Screening of resistant recombinant yeast (A) and PCR identification results (B);

[0028] Figure 9 The results of SDS-PAGE (A) and WB (B) identification of the recombinant protein;

[0029] Figure 10 Schematic diagram of the recombinant protein structure;

[0030] Figure 11 SDS-PAGE (A) and growth curve (B) yield analysis of fermentation products at different times;

[0031] Figure 12 SDS-PAGE of the fermentation product in BSM medium (A) and its purified product (B). DETAILED DESCRIPTION

[0032] Test strains and cells

[0033] Gram-negative bacteria: Escherichia coli ATCC29522, Salmonella gullinarum ATCC10398;

[0034] Gram-positive bacteria: Staphylococcus aureus (ATCC 43300), Clostridium perfringens (ATCC 13124), Streptococcus agalactiae (ATCC 13813), and Streptococcus faecalis (ATCC 29212);

[0035] Mouse breast cancer cells 4T1.2 (RPMI Medium 1640 culture medium), human lung cancer cells A549 (DMEM culture medium), HeLa cells (DMEM culture medium), human breast cancer cells MCF-7 (DMEM culture medium), and human breast cancer cells MDA-MB-231 (RPMI Medium 1640 culture medium);

[0036] Escherichia coli DH5α containing the pGAPZαA plasmid and Pichia pastoris host strain SMD1168.

[0037] Culture medium and test reagents

[0038] MH broth (MHB), MH agar (MHA), Dulbecco's Modified Eagle's Medium (DMEM) cell culture medium (Hyclone), fetal bovine serum (Hyclone), trypsin (Gibco), RPMI Medium 1640, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride, potassium chloride, concentrated hydrochloric acid, Triton X-100, yeast extract powder, beef powder, soluble starch, and acid-hydrolyzed casein;

[0039] Tryptone, peptone, yeast extract, sodium chloride, sorbitol, sodium acetate trihydrate, 1 kb ladder, XbaⅠ, EcoRⅠ, BlnⅠ, bleomycin, plasmid DNA minipreparation kit, and yeast genomic DNA extraction kit.

[0040] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1: Synthesis and preservation of antimicrobial peptides

[0042] The sequence of the designed antimicrobial peptide CC28 (SEQ ID NO. 1: GWLKKIKKIHRVQHRAIHPILKLIGGLL) was sent to Jier Biochemical (Shanghai) Co., Ltd. for chemical synthesis (solid-phase synthesis using Fmoc protection). After synthesis, it was purified by high-performance liquid chromatography to a purity of over 90%, and peptide mass spectrometry (MALDI-TOF MS) was performed to detect the molecular mass and determine whether the sequence was successful. The peptide mass spectrometry results and the CC28 structure diagram are shown in FIG. Figure 1 and Figure 2 As shown, it shows that the novel antimicrobial peptide CC28 of the present invention was successfully synthesized and stored at -80°C.

[0043] Determination of minimum inhibitory concentration (MIC) of antimicrobial peptide CC28

[0044] 1. The minimum inhibitory concentration was determined using a 96-well plate method: the bacterial solution stored at -80°C was taken out and streaked on a plate, and a single colony was picked and cultured in the corresponding liquid culture medium. The test bacteria were cultured at 37°C with air shaking at 200r / min until the logarithmic growth phase of the bacteria, with OD600≈0.6. The synthesized antimicrobial peptide was graded diluted with MHB culture medium to: 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5μg / mL. The cultured bacterial solution was diluted to 10 5CFU / mL, and 50 μL per well of a 96-well culture plate was added. Then, 50 μL of the antimicrobial peptide diluted to a fixed concentration was added to each well. Three replicates were set for each sample, with culture medium alone as a negative control and test bacteria alone as a positive control. Cultures were performed for 20-24 hours. The absorbance was measured at 625 nm using a microplate reader. The concentration immediately preceding the sudden increase in absorbance was the minimum inhibitory concentration (MIC) of the test bacteria.

[0045] Test bacteria: Escherichia coli ATCC 29522, Salmonella gullinarum ATCC 10398, Clostridium perfringens ATCC 13124, Streptococcus faecium ATCC 29212, Streptococcus agalactiae ATCC 13813, and Staphylococcus aureus ATCC 29522.

[0046] The results showed that CC28 had a strong inhibitory effect on Escherichia coli, Clostridium perfringens and Streptococcus agalactiae, as shown in Table 1 below:

[0047] Table 1 Minimum inhibitory concentration (MIC, μmol / L) of antimicrobial peptides

[0048]

[0049]

[0050] 2. Minimum bactericidal concentration (MBC) determination

[0051] All antimicrobial peptide concentrations before the MIC value are mixed with the test bacteria and then spread on the solid culture medium. The plate where the test bacteria do not grow is the antimicrobial peptide concentration used.

[0052] From the bactericidal results of CC28 in Table 2, it can be seen that the bactericidal effect of this antimicrobial peptide on Escherichia coli is relatively stable.

[0053] Table 2 Minimum bactericidal concentration (MBC, μmol / L) of antimicrobial peptides

[0054]

[0055] 3. Hemolytic activity detection

[0056] Collect 5 ml each of sheep, rabbit, rat, and adult male (human) blood and centrifuge at 1500 × g for 5 minutes. Discard the upper serum layer and collect the lower red blood cells. Wash three times with PBS and centrifuge three times at 1500 × g for 5 minutes each to prepare a 2% fresh red blood cell suspension. Add 50 μL of the red blood cell suspension to wells 1 through 10 of each row of a 96-well plate, and 25 μL to wells 11 and 12. Add 50 μL of 512 μg / L antimicrobial peptide to well 1, mix thoroughly, and then pipette 50 μL into well 2, then to well 10. Remove 50 μL from well 10 and discard. Add 25 μL of PBS to well 11 and 25 μL of Triton-100 to well 12. Incubate at 37°C for 1 hour, and measure absorbance at 570 nm. Sterile PBS and 0.1% (v / v) Triton-100 were used as standards for hemolysis percentages of 0% and 100%. Three replicates were performed for each sample, and the average value was calculated. (Hemolysis rate (%) = (sample absorbance - saline control absorbance) / (0.1% Triton-100 control absorbance - saline control absorbance) * 100%). Table 3 shows that the antimicrobial peptide had low hemolytic activity against rabbits and sheep.

[0057] Table 3 Antimicrobial peptide 5% hemolysis concentration (μmol / L)

[0058] antimicrobial peptides people mouse rabbit sheep CC28 4.4 2.2 8.9 7.26

[0059] The therapeutic index of each antimicrobial peptide was obtained by dividing the average MIC value of the antimicrobial peptide against harmful bacteria by the 5% hemolysis concentration. The results are shown in Table 4. As shown in the table, the therapeutic index of antimicrobial peptide CC28 is high for rabbits and humans.

[0060] Table 4 Therapeutic index (TI) of antimicrobial peptides

[0061] antimicrobial peptides people mouse rabbit sheep CC28 0.47 0.23 0.93 0.23

[0062] 4. Detection of anti-tumor cell activity of antimicrobial peptides

[0063] a. Cell Thawing: Wearing sterile gloves, remove the cryopreserved tube of cells from the liquid nitrogen tank. Quickly place the tube in a 37°C water bath, shaking occasionally, and completely thaw within 1 minute. Then, remove the cells under sterile conditions. Add 5 mL of culture medium and inoculate the tube into a culture flask. Incubate the tube in a 37°C incubator. Change the culture medium once the next day, continue culturing, and observe growth.

[0064] b: Cell passage: When the cell fusion reaches 90%, discard the 25cm 2Wash the cells in the culture flask twice with PBS; add 1.5 mL of 0.25% trypsin digestion solution and incubate in a 37°C incubator for a specified period of time. Once the cells have shrunk and become rounded, add complete culture medium to terminate digestion. Gently pipette the cells to dislodge them, transfer the cell suspension to a 15 mL centrifuge tube, and centrifuge at 1.000 g for 5 minutes. Discard the supernatant and resuspend the cell pellet in fresh culture medium. Adjust the cell ratio to a 1:2-1:4 ratio based on the cell proliferation rate for passage. Change the cell culture medium every two days. Fill in the corresponding experimental record for each observation, and implement the principle of recording individual cells separately.

[0065] c: Cell plating: Obtain a certain amount of cell pellet and resuspend it in complete culture medium to adjust the cell concentration to 1×10 6 Each concentration gradient was plated in 96-well plates with 8 replicate wells, 100 μL per well, and 8 replicate wells per well.

[0066] d: Cell dosing: The protein solution was replaced after 24 hours. Before the experiment, the protein solution was diluted with the basal culture medium corresponding to the cells. The dilution gradient was 50 μg / mL (a), 100 μg / mL (b), 150 μg / mL (c), 200 μg / mL (d), and basal culture medium wells. The cell status was observed 24 hours after dosing, and pictures of cell growth under a microscope at each protein concentration were taken using a mobile phone. Cell viability was then detected using CCK8.

[0067] e: Cell viability assay: Prepare 10% CCK8 assay solution in the same basal medium used for the cells. Wash a 96-well plate with HBSS and pat dry any remaining liquid on absorbent paper. Add 100 μL of CCK8 assay solution to each well in the dark, setting up calibration wells. Incubate in a light-proof incubator at 37°C. Measure the OD value on a microplate reader every 30 minutes or every hour depending on the cell density. Stop the assay when the OD value of the basal medium reaches a range of 0.7 to 1.0. Calculate the percentage of cell viability at each concentration gradient using the obtained OD values.

[0068] In this example, the test cells are: mouse breast cancer cells 4T1.2 (such as Figure 3 ), HeLa cells ( Figure 4 ), human breast cancer cell line MCF-7 ( Figure 5 ), human breast cancer cells MDA-MB-231 ( Figure 6 ), the results showed that the antimicrobial peptide CC28 had a strong inhibitory effect on three breast cancer cells among the four tested reproductive cancer cells, with the concentration range of 13.3μmol / L to 43.5μmol / L. These experimental results indicate that CC28 has good anti-tumor activity.

[0069] Example 2: Protein and Gene Design of CC28 Fusion The amino acid sequence of CC28-cellulase protein is shown in SEQ ID NO.2:

[0070] EFHHHHHHDDDDKGWLKKIKKIHRVQHRAIHPILKLIGGLLEAAAKEAAAKEAAAKGGGGSGGGGSMNKPMGPLLLATALMASGAIAQTQSVWGQCGGTGYSGPTNCASGSACSTLNPYYA QCIPGATSFITSTTSTKSPGSGSSTTSSASQPTGSGQTRFAGINIAGFDFGCTIDGTCVTSQIPYPLKNFGGTNNHPDGVGQMQHFVNDDKLNIFRLVPGWQYLVNNNLGGTLDSTAISNY DQLVQGCLATGSYCIVDIHNYARWNGAIIGQGGPTNAQFVSLWTQLATKYASQSVKWFGIVNEPHDVDINTWGQTVQAVVTAIRNAGATTQFISLPGTDFQSAGSFLSDGSSTALSQVKNP DGSTTNLIFDFKHYLDSDNSGTHTECVTNNIATFAQPVATWLRQNKRQGILTETGGGNTQSCLTDMCQQNQFLNQNSDVFLYGIGWGAGSFDSTYELTLTPTQNGNTWTDTALAAACFSR.

[0071] The design process is as follows:

[0072] The amino acid sequence of the antimicrobial peptide in this design is numbered as SEQ ID NO.1, and the amino acid sequence of the linker design is SEQ ID NO.7: (EAAAK)3(GGGGS)2;

[0073] Reference is made to the description of [Chen, X.; Bai, Y.; Zaro, JL; Shen, WC, Design of an in vivo cleavable disulfide linker in recombinant fusion proteins. BioTechniques 2010, 49(1), 513-8. DOI: 10.2144 / 000113450.]; the sequence of ligand XX (SEQ ID NO. XP_018657255.1) was retrieved through NCBI, and its AA sequence is: MNKPMGPLLLAATLMASGAIAQTQSVWGQCGGTGYSGPTNCASGSACSTLNPYYAQCIPGATSFITSTTSTKSPGSGSSTTSSASQPTGSGQTRFAGINIAGFDFGCTIDGTCVTSQIYPPLKNFGGTNNHPDGVGQMQHFVNDDKLNIFRLPVGWQYLVNNNLGGTL DSTAISNYDQLVQGCLATGSYCIVDIHNYARWNGAIIGQGGPTNAQFVSLWTQLATKYASQSKVWFGIVNEPHDVDINTWGQTVQAVVTAIRNAGATTQFISLPGTDFQSAGSFLSDGSSTALSQVK NPDGSTTNLIFDFHKYLDSDNSGTHTECVTNNIATAFQPVATWLRQNKRQGILTETGGGNTQSCLTDMCQQNQFLNQNSDVFLGYIGWGAGSFDSTYELTLTPTQNGNTWTDTALAAACFSR, is SEQ ID NO.8;

[0074] The CC28 gene sequence is coded as SEQ ID NO. 3, 123 bp, sequence:

[0075] GAATTC CACCACCACCACCACCATGATGATGATGATAAGGGTTGGTTGAAGAAGATT

[0076] AAGAAGATTCACAGAGTCCAACACAGAGCTATCCACCCAATTTTGAAGTTGATTGG TGGTTTGTTG;

[0077] The linker sequence is encoded as SEQ ID NO. 4, 75 bp, and the sequence is:

[0078] GAAGCTGCCGCTAAGGAAGCCGCTGCTAAGGAAGCCGCTGCTAAGGGTGGTGGTGGTTCTGGTGGTGGTGGATCC;

[0079] The ligand sequence is encoded as SEQ ID NO.5 and the sequence is:

[0080] ATGAACAAGCCAATGGGTCCTTTGCTGTTGGCTGCTACCTTGATGGCTTCTGGTGCC

[0081] ATTGCTCAAACCCAATCTGTTTGGGGTCAATGTGGTGGTACTGGTTACTCCGGTCCA

[0082] ACTAACTGTGCTTCTGGATCTGCTTGTTCTACTTTGAACCCATACTACGCTCAATGTA

[0083] TTCCAGGTGCTACTTCTTTCATTACTTCCACCACCTCCACTAAGTCCCCAGGATCCG

[0084] GATCCTCTACCACTTCTTCCGCTTCTCAACCAACCGGTTCCGGTCAGACTAGATTCG

[0085] CTGGTATTAACATCGCTGGTTTCGACTTCGGATGTACTATTGATGGTACTTGTGTTAC

[0086] TTCCCAAATCTACCCACCACTTAAGAACTTCGGAGGTACTAACAACCACCCAGACG

[0087] GAGTTGGTCAAATGCAACACTTCGTTAACGATGATAAGTTGAACATTTTCAGATTGC

[0088] CAGTTGGTTGGCAATACTTGGTTAACAACAACTTGGGTGGTACTTTGGATTCTACTG

[0089] CTATTTCTAACTACGACCAACTGGTTCAAGGTTGTTTGGCTACTGGATCTTACTGTAT

[0090] CGTTGACATTCACAACTACGCTAGATGGAACGGTGCTATCATCGGACAAGGTGGTCC

[0091] AACTAACGCTCAGTTCGTTTCCCTGTGGACTCAATTGGCTACTAAGTACGCTTCCCA

[0092] ATCTAAGGTTTGGTTTGGTATTGTTAATGAGCCTCATGATGTTGACATTAATACTTGG

[0093] GGTCAAACCGTCCAGGCTGTTGTTACTGCTATTCGTAACGCTGGTGCCACTACTCAA

[0094] TTCATCTCCTTGCCAGGTACTGACTTCCAATCTGCTGGTTCTTTCCTTTCTGATGGAT

[0095] CCTCTACCGCTTTGTCCCAAGTTAAGAACCCTGATGGTTCTACTACTAACTTGATCTT

[0096] CGATTTCCACAAGTACTTGGATTCTGATAACTCTGGTACTCATACTGAATGTGTTACT

[0097] AACAACATCGCTACCGCTTTCCAACCAGTTGCTACTTGGTTGAGACAGAACAAGAG

[0098] ACAAGGAATCCTGACTGAAACTGGTGGTGGTAACACTCAATCTTGTTTGACTGATAT

[0099] GTGTCAACAAAACCAATTCTTGAATCAAAACTCTGATGTTTTCCTGGGTTACATTGG

[0100] TTGGGGTGCTGGTTCTTTCGATTCTACTTACGAATTGACTTTGACTCCAACTCAAAA

[0101] CGGTAACACTTGGACTGATACCGCTTTGGCTGCTGCTTGTTTCTCCAGAAAG TCTAGA ;

[0102] The target protein gene sequence is encoded as SEQ ID NO.6, 1458bp, and the sequence is:

[0103] GAATTC CACCACCACCACCACCATGATGATGATGATAAGGGTTGGTTGAAGAAGATT

[0104] AAGAAGATTCACAGAGTCCAACACAGAGCTATCCACCCAATTTTGAAGTTGATTGG

[0105] TGGTTTGTTGGAAGCTGCCGCTAAGGAAGCCGCTGCTAAGGAAGCCGCTGCTAAGG

[0106] GTGGTGGTGGTTCTGGTGGTGGTGGATCCATGAACAAGCCAATGGGTCCTTTGCTG

[0107] TTGGCTGCTACCTTGATGGCTTCTGGTGCCATTGCTCAAACCCAATCTGTTTGGGGT

[0108] CAATGTGGTGGTACTGGTTACTCCGGTCCAACTAACTGTGCTTCTGGATCTGCTTGT

[0109] TCTACTTTGAACCCATACTACGCTCAATGTATTCCAGGTGCTACTTCTTTCATTACTTC

[0110] CACCACCTCCACTAAGTCCCCAGGATCCGGATCCTCTACCACTTCTTCCGCTTCTCA

[0111] ACCAACCGGTTCCGGTCAGACTAGATTCGCTGGTATTAACATCGCTGGTTTCGACTT

[0112] CGGATGTACTATTGATGGTACTTGTGTTACTTCCCAAATCTACCCACCACTTAAGAAC

[0113] TTCGGAGGTACTAACAACCACCCAGACGGAGTTGGTCAAATGCAACACTTCGTTAA

[0114] CGATGATAAGTTGAACATTTTCAGATTGCCAGTTGGTTGGCAATACTTGGTTAACAA

[0115] CAACTTGGGTGGTACTTTGGATTCTACTGCTATTTCTAACTACGACCAACTGGTTCA

[0116] AGGTTGTTTGGCTACTGGATCTTACTGTATCGTTGACATTCACAACTACGCTAGATGG

[0117] AACGGTGCTATCATCGGACAAGGTGGTCCAACTAACGCTCAGTTCGTTTCCCTGTG

[0118] GACTCAATTGGCTACTAAGTACGCTTCCCAATCTAAGGTTTGGTTTGGTATTGTTAAT

[0119] GAGCCTCATGATGTTGACATTAATACTTGGGGTCAAACCGTCCAGGCTGTTGTTACT

[0120] GCTATTCGTAACGCTGGTGCCACTACTCAATTCATCTCCTTGCCAGGTACTGACTTCC

[0121] AATCTGCTGGTTCTTTCCTTTCTGATGGATCCTCTACCGCTTTGTCCCAAGTTAAGAA

[0122] CCCTGATGGTTCTACTACTAACTTGATCTTCGATTTCCACAAGTACTTGGATTCTGAT

[0123] AACTCTGGTACTCATACTGAATGTGTTACTAACAACATCGCTACCGCTTTCCAACCA

[0124] GTTGCTACTTGGTTGAGACAGAACAAGAGACAAGGAATCCTGACTGAAACTGGTG

[0125] GTGGTAACACTCAATCTTGTTTGACTGATATGTGTCAACAAAACCAATTCTTGAATC

[0126] AAAACTCTGATGTTTTCCTGGGTTACATTGGTTGGGGTGCTGGTTCTTTCGATTCTAC

[0127] TTACGAATTGACTTTGACTCCAACTCAAAACGGTAACACTTGGACTGATACCGCTTTGGCTGCTGCTTGTTTCTCCAGAAAG TCTAGA. The marker codes GAATTC and TCTAGA are the restriction sites of EcoRⅠ and XbaⅠ, respectively. GATGATGATGATAAG The sequence is His tag and protease cleavage site.

[0128] The designed gene sequence SEQ ID NO.6 was sent to Sangon Biotech (Shanghai) Co., Ltd. for full gene synthesis and subcloning into the pGAPZαA expression vector. The recombinant expression vector was then maintained in E. coli XL-10. Example 3: Identification and transformation of pGAPZαA / CC28

[0129] 1. Double enzyme digestion identification of recombinant expression vector: Take out the E. coli DH5α containing the recombinant expression vector pGAPZαA / CC28 stored in a -80℃ refrigerator, streak inoculate on low-salt LB solid medium containing 25μg / mL Zeocin resistance, and culture at 37℃ overnight. Select a single colony with good growth and inoculate it into a 10mL test tube containing 5mL low-salt LB medium containing 25μg / mL Zeocin resistance after high temperature sterilization, and culture it at 37℃ with air shaking at 220r / min overnight. Use AxyPrep Plasmid DNA Mini Extraction Kit to extract the plasmid. Refer to the kit manual for the experimental steps and perform double enzyme digestion according to the specified system. The results are as follows Figure 7 A, After double enzyme digestion, the recombinant expression vector pGAPZαA / CC28-cellulase obtained the target gene fragment at 1458 bp in lanes 1 and 2, and a vector molecular band was found at nearly 3000 bp, indicating that the vector was constructed correctly.

[0130] 2. PCR identification of recombinant pGAPZαA / CC28: Detection was performed using the recombinant plasmid pGAPZαA / CC28-cellulase as a template, and PCR verification was performed using universal primers for pGAPZαA. The recombinant plasmid PCR reaction system used was operated according to the instructions of the kit.

[0131] Recombinant plasmid DNA extracted from Escherichia coli XL-10 was linearized with the restriction endonuclease Bln I. DNA was extracted from the bacterial suspension using a yeast genomic DNA extraction kit. PCR was performed using yeast genomic DNA as a template and the pGAPZαA universal vector primers α-FACTOR and 3'AOXI to verify integration of the recombinant plasmid into the Pichia pastoris genome.

[0132] Primer: 3'AOX1:GGCAAATGGCATTCTGACATCCTC (SEQ ID NO. 9)

[0133] α-FACTOR:TACTATTGCCAGCATTGCTGC(SEQ ID NO.10)

[0134] The recombinant bacterial solution identified as positive was sent to Harbin Borui Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the designed target gene using DNAMAN. The results are as follows Figure 7 B. After PCR using the obtained primers, a bright specific band appeared at 1611 bp in the recombinant expression vector pGAPZαA / CC28-cellulase, further demonstrating the successful construction of the recombinant vector at the gene level and indicating that the recombinant plasmid was successfully integrated into the Pichia pastoris genome.

[0135] 3. Preparation of competent state of Pichia pastoris: Take out the yeast stored in -80℃ refrigerator, streak culture on YPD solid medium, and then culture the yeast in 30℃ constant temperature biochemical incubator for 2 days until a single colony is formed (this process ensures that there are no foreign bacteria, and the plate mouth can be covered with sealing film). Take a single colony and inoculate it into a 5mL YPD test tube, culture it at 30℃ 220r / min overnight for about 16h, take 800μL of mature bacterial solution and inoculate it into 50mL YPD medium, 220r / min, 30℃ air shaking culture, and culture it until the bacterial solution concentration is OD 600=0.8~1.0 (this process is about 8 hours). The bacterial solution is transferred to a 50mL centrifuge tube, 5000r / min, 4 ℃ centrifugal 10min and the supernatant is discarded. 25mL of sterilized deionized water is added to the precipitated cells to resuspend the yeast cells in deionized water at 5000r / min, 4 ℃ centrifugal 10min and the supernatant is discarded. The precipitated cells are recovered, 10mL of sterilized deionized water is added to wash the cells once and suspend the cells at 5000r / min, 4 ℃ centrifugal 10min, 1mL100mM LiCl solution is added to the collected cells, and the remade cell suspension is moved to a 1.5mL centrifuge tube, 14000r / min centrifugal 1min to precipitate the bacteria, and carefully remove the 100mM LiCl solution in the centrifuge tube, then add 400μL 100mM LiCl solution to the centrifuge tube, and aliquot according to 50μL per tube for immediate transformation.

[0136] 4. Linearization and transformation of pGAPZαA / CC28: The positive recombinant pGAPZαA / CC28 stored at -80℃ was expanded, and then re-extracted according to the instructions of the small amount of plasmid DNA extraction reagent to determine its concentration. The recombinant plasmid was completely linearized with AvrⅡ linearization enzyme (TaKaRa), and then recovered and concentrated to obtain linear plasmid DNA for detection. Figure 7 As shown in C, after being treated with the linearization enzyme, an obvious band appeared at 4558 bp in the recombinant expression vector pGAPZαA / CC28-cellulase, indicating that the recombinant plasmid was completely linearized.

[0137] Mix 80uL of yeast competent cells with 10μg of linearized plasmid in an ice bath, then transfer to a 2mm pre-cooled electroporation cuvette and place on ice for 5 minutes before starting electroporation (electroporation conditions: voltage, 275V; pulse length: 15ms; number of pulses: 3 times; capacitance, 25μF; resistance, 200Ω). After the electroporation is completed, immediately add the pre-cooled 1M sorbitol solution to the 2mm electroporation cuvette and transfer the mixture to a 1.5mL EP tube. The mixed product is activated at 30℃ for 2h. The activated bacterial solution is spread on YPDS solid culture medium with 100μg / mL Zeocin, and then inverted and placed in a 30℃ constant temperature biochemical incubator for 3-5 days until a single colony grows ( Figure 8 A).

[0138] 5. Cultivation of positive transformants: Select single colonies with good growth after transformation and re-spread them on YPDS solid medium containing 100 μg / mL Zeocin. Select single colonies with good growth and inoculate them into 5 mL of YPDS liquid medium containing 100 μg / mL Zeocin. Cultivate at 30°C, 220 rpm and shaking for 24 h.

[0139] 6. Yeast genomic DNA extraction and PCR identification: DNA was extracted from the bacterial suspension using a yeast genomic DNA extraction kit (Solabo). The operating steps were referred to the kit instructions.

[0140] Using yeast genomic DNA as a PCR template, PCR was performed using the pGAPZαA universal vector primers α-FACTOR and 3'AOXI to identify whether the recombinant plasmid was integrated into the Pichia pastoris genome. The extracted yeast DNA was sequenced to identify whether the gene matched the design. The identification results were as follows: Figure 8 B, indicating that the target gene has been successfully transferred into Pichia pastoris.

[0141] Example 4 Expression, purification and activity detection of pGAPZαA / CC28-cellulase gene in Pichia pastoris

[0142] 1. Protein concentration: Take 1 mL of recombinant Pichia pastoris engineered CC28-cellulase culture medium and inoculate it into 100 mL of YPD liquid medium for expansion. After a period of incubation, centrifuge at 12,000 rpm for 2 minutes. Transfer the supernatant of the centrifuged culture liquid to a centrifuge tube for preliminary protein concentration. Take 1 mL of the culture liquid in a 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 1 minute at 4°C. Take 500 μL of the supernatant of the culture liquid and centrifuge at 12,000 rpm for 3 minutes at 4°C. Add anhydrous ethanol, mix well, and place at -20°C for more than 1 hour. Centrifuge and discard the supernatant. Rinse the precipitate with 75% ethanol and centrifuge again to discard the supernatant. Repeat the above steps, dry the precipitate naturally, and dissolve it in 35 μL of water.

[0143] 2. Protein Concentration Determination: Use a BCA kit to determine the protein content in the supernatant of the recombinant Pichia pastoris CC28-cellulase culture. First, prepare a protein standard in a gradient concentration. Measure the absorbance corresponding to different concentrations using a microplate reader. Use Excel 2010 to plot a protein standard curve, derive a regression equation, and calculate the protein concentration of the sample based on this equation.

[0144] 3. SDS-PAGE detection of target protein: prepare separation gel and stacking gel, take protein sample and protein loading buffer and mix them evenly, then boil in water bath for 10 minutes, take sample and load, electrophoresis at 60V for 30 minutes, after entering separation gel, electrophoresis at 120V for 1 hour, after the electrophoresis, fix the separation gel for 40 minutes, stain for 30 minutes, decolorize overnight, and use gel imaging system to observe the gel. The results are as follows: Figure 9 A.

[0145] 4. Western blot detection of target protein: transfer the gel to PVDF membrane and block the membrane for 2 hours. Then, use His-tag antibody (mouse monoclonal antibody) as primary antibody and incubate at 4°C overnight. Use horseradish peroxidase-labeled goat anti-mouse IgG (H+L) as secondary antibody (Biyuntian) and incubate at low speed for 50 minutes. After that, use ECL to develop the PVDF membrane and place it on the gel imaging system for exposure and imaging. Save and analyze the results. Figure 9 B and Figure 10 shown.

[0146] Depend on Figure 9 It can be seen that the expression product of the recombinant bacteria can specifically bind to His-tag (mouse monoclonal antibody), and a specific reaction band appears at 51.43 KDa, which proves that the antimicrobial peptide CC28 is expressed in Pichia pastoris SMD1168.

[0147] Example 5 YPD medium fermentation of CC28-cellulase protein secretion pattern of yeast engineering bacteria

[0148] Take the recombinant Pichia pastoris CC28-cellulase seed solution and transfer it to 100 mL of YPD medium and BSM medium at a 5% inoculum volume, respectively. Cultivate at 220 r / min and 30°C with shaking. Take the culture solution every 12 h according to the marked cultivation time sequence for protein concentration, BCA concentration determination and SDS-PAGE identification (see Figure 11 A), analyze the target band ratio by gel software to calculate the target protein yield in YPD at different time periods. Draw the target protein yield curve with the target protein yield (mg / mL) as the vertical axis and the culture time (h) as the horizontal axis, as shown in Figure 11 B.

[0149] Depend on Figure 11 It can be seen that the yield of the target protein in YPD changes with time. The target protein content gradually increases in the first 24 hours, and the impurity protein is relatively small. The target protein yield reaches the highest at 24 hours, which is 4.22 mg / mL. After 24 hours of culture, as the culture time increases, the impurity protein content increases, and the target protein content gradually decreases.

[0150] Example 6 Purification of the target protein of pGAPZαA / CC28-cellulase:

[0151] (1) Protein concentration: 100 mL of recombinant Pichia pastoris CC28-cellulase culture medium cultured for 24 h in BSM medium (the culture method is the same as the method for fermenting CC28-cellulase protein in YPD medium as described above) was taken and centrifuged at 4°C, 14,000 r / min, for 30 min. The ground solid ammonium sulfate powder was slowly added to the supernatant of the centrifuged culture medium to achieve a saturation of 80%. The supernatant was stirred at 4°C for 2 h and then centrifuged at 4°C, 10,000 r / min, for 30 min. The resulting precipitate was collected and dissolved in 10 mL of PBS. The precipitate was placed in a treated dialysis bag and dialyzed at 4°C for 50 h. The dialysate was replaced every 4 h to avoid ammonium sulfate residue in the protein.

[0152] (2) Pretreatment of nickel column: The storage liquid of affinity medium pre-packed column (Zhongke Senhui) is 20% ethanol. After all materials and reagents reach the same temperature, before use, it is necessary to wash away the 20% ethanol with deionized water, washing with water for about 5 column volumes.

[0153] (3) Equilibrate the chromatography column with 10 column volumes of equilibration buffer until the conductivity and pH value of the effluent remain unchanged.

[0154] (4) Sample loading: Place the column in an ice bath. Wait until the equilibration buffer flows to 2 mm above the packing. Use a pipette to load the sample. To avoid bubbles, drain the sample through a glass cup or add it along the inner wall of the column to minimize excessive dilution. Collect the discharged flow-through into a 10 mL centrifuge tube. Repeat the loading several times and collect the sample.

[0155] (5) Wash the impurities with a gradient elution buffer. Each gradient elution buffer is 5 times the column volume. Collect the rinse solution in a 10 mL centrifuge tube.

[0156] (6) Elution: Use 10 column volumes of 500 mM imidazole eluent, pH = 7.4 to rinse the target protein, and collect the eluent.

[0157] (7) Storage: Rinse the chromatography column thoroughly with pure water and store it in 20% ethanol at 4°C.

[0158] (8) Treatment of protein purification solution: The collected protein purification solution was subjected to SDS-PAGE electrophoresis identification, and the remaining purified protein liquid was vacuum freeze-dried and stored. The results are shown in Figure 12 , Figure 12 A shows the results of BSM fermentation (lanes 1 and 2 are both rCC28-cel (near 50 kDa)); Figure 12B shows the purification results (lanes 1-5: 1: pre-column, 2: 500 mM imidazole elution, 3: 60 mM imidazole elution, 4: 40 mM imidazole elution, 5: flow-through). The antimicrobial peptide CC28-coupled cellulase complex showed a distinct band at 51.43 kDa, with no other contaminants, demonstrating successful purification of the antimicrobial peptide.

[0159] In addition, in conjunction with Example 5, the total protein concentration in the supernatant was determined by BCA, and it was found that the total protein concentration in the YPD supernatant was 4.71 mg / mL, while the total protein concentration in the BSM supernatant in Example 6 was 10.19 mg / mL. According to the proportion of the target band, it was calculated that the target protein content in the YPD culture medium was 4.14 mg / mL, and the target protein content in the BSM culture was 6.7 mg / mL. Therefore, under the same 24-hour culture conditions, the BSM culture medium obtained a high target protein content and low cost, which is more conducive to subsequent large-scale fermentation and air-drying spray powder production.

[0160] Example 7: Antibacterial activity analysis of CC28-cellulase expression protein

[0161] This experiment uses the inhibition zone method to detect the antibacterial activity of the purified product against bacteria. Among them, Gram-negative bacteria include Escherichia coli, Salmonella enterica, and Pseudomonas aeruginosa, and Gram-positive bacteria include Streptococcus faecalis, Staphylococcus aureus, and Streptococcus dysgalactiae. The above strains were streaked on MHA solid culture medium. After a single colony grew, a single colony was picked up with an inoculation loop and placed in 5 mL MHB culture medium. The culture was shaken at 37°C and 220 r / min until the bacterial suspension of each strain grew for 10 5 -10 8 cfu / mL and take out for later use.

[0162] 100 μL of each bacterial solution was spread onto a plate using the plate-spreading method. Sterilized filter paper was evenly placed on the plate, and 20 μL of the purified antimicrobial peptide target protein solution was added to each filter paper. The plates were incubated at 37°C for 24 hours. Preliminary antibacterial activity was assessed by observing the size of the inhibition zone. As shown in Table 5, the purified product exhibited antibacterial activity against Escherichia coli and Staphylococcus aureus, but not against other harmful bacteria.

[0163] Table 5 Inhibition zone radius of recombinant antimicrobial peptides (mm)

[0164]

[0165] Example 8: Determination of Cellulase Activity of pGAPZαA / CC28-Cellulase Expression Protein Solution

[0166] CMC-Na enzyme activity assay

[0167] 1. Enzyme solution to be tested: Aseptically pipette 5 mL of bacterial solution from a shake flask and perform gradient dilutions;

[0168] 2. Determination: Take a 25 mL stoppered test tube and add 1.5 mL of CMC-Na solution and 0.5 mL of appropriately diluted enzyme solution in sequence. After shaking, place in a 50°C constant temperature water bath for hydrolysis for 30 min. Immediately add 1.5 mL of DNS color developer and boil in a boiling water bath for 5 min. Immediately cool to room temperature with running water, dilute to volume with distilled water, and measure the absorbance As at 520 nm.

[0169] 3. Blank sample: Centrifuge the bacterial solution to be tested and sterilize it at 121°C under high temperature and high pressure. Then, perform the same steps as in step 2 on the treated bacterial solution and measure the absorbance Ack at 520 nm.

[0170] △A=As-Ack According to △A, the glucose content P is obtained from the standard curve;

[0171] 4. Calculation

[0172]

[0173] Note: U represents enzyme activity; P represents glucose content P found on the standard curve; K represents enzyme solution dilution multiple; T represents constant temperature water bath time.

[0174] Filter paper enzyme activity assay

[0175] (1) Take 0.5 mL of appropriately diluted enzyme solution and add 1 mL of 0.1 mol / L acetic acid-sodium acetate buffer with a pH of 4.8;

[0176] (2) Add 50±0.5mg of filter paper (1cm×6cm) and heat the enzymatic reaction at 50℃ for 1 hour (preheat for 5 minutes)

[0177] (3) Add 3 mL of DNS colorimetric solution (1.5 mL for standard curve), place in boiling water for 10 min, cool in running water, and measure absorbance at 520 nm.

[0178] (4) At the same time, use enzyme solution sterilized at 121°C (refer to CMC-Na enzyme activity assay blank sample) as a control to subtract background;

[0179] (5) According to the absorbance, find the corresponding glucose content from the glucose standard curve, and calculate the enzyme activity based on the number of grams of glucose generated. Filter paper enzyme activity is calculated according to the following formula:

[0180]

[0181] Note: U represents enzyme activity; P represents glucose content P found on the standard curve; K represents enzyme solution dilution multiple; T represents constant temperature water bath time.

[0182] The results showed that the molecular weight of the target protein of recombinant Pichia pastoris pGAPZαA / CC28-cellulase / SMD1168 was 51.43 KDa;

[0183] Table 6 Recombinant engineering bacteria CC28-cellulase activity (U / L)

[0184]

[0185] As shown in Table 6, the recombinant Pichia pastoris pGAPZαA / CC28-cellulase / SMD1168 engineering bacterial solution had cellulase activity, with the filter paper enzyme activity being 720 U / L and the sodium carboxymethyl cellulose enzyme activity being 1820 U / L.

[0186] These experimental results show that the pGAPZαA / CC28-cellulase / SMD1168 engineered bacterial solution can successfully express the target protein with high protein yield and both antibacterial activity and cellulase activity.

[0187] Example 9 Broiler Diet Composition and Feeding Management

[0188] The diet used was a corn-soybean meal feed, formulated with reference to the GB / T 5916-2020 chicken husbandry standard (see Table 7). The experiment used a caged chicken house model, with humidity controlled at 60% to 70%. The temperature was maintained at 35-37°C for the first three days of the experiment, then decreased by 0.5°C daily, maintaining a stable temperature from the fourth day of age. During the first week of the experiment, the light was on for 24 hours, and then the light duration was gradually shortened until 16 hours of light could be maintained, at which point the light duration was no longer shortened. The chicks were vaccinated with Newcastle disease and bursal disease vaccines according to normal immunization procedures. The temperature and humidity of the chicken house were recorded throughout the experiment, and proper ventilation was maintained. Regular cleaning and disinfection were also carried out.

[0189] Table 7 Diet composition and nutritional levels

[0190]

[0191]

[0192] Example 10: Determination of growth performance

[0193] Experimental animals and group treatment

[0194] This study used a single-factor, completely randomized design. Two hundred broiler chickens in good condition and of similar body weight were randomly divided into two groups: a control group (CON) and a CC28-cellulase 5g group (CC28×5g). The CON group was fed a basal diet, while the CC28×5g group received a basal diet supplemented with 5g / kg of the antimicrobial peptide CC28-cellulase powder. Each gram of engineered bacterial powder contained 41.59 mg of the target protein. Each group had four replicates, 25 birds per replicate, with free access to water and standard care. The experimental period lasted 42 days. Birth weight was measured for each group, and the amount of spread feed was recorded regularly. The birds were weighed at the end of each week. At the end of the experiment, average daily gain (ADG), average daily feed intake (ADFI), and feed-to-meat ratio (F / G) were calculated based on body weight and spread feed. The results are shown in Table 8.

[0195] Calculation formula: average daily weight gain = (final weight - initial weight) / days; average daily feed intake = feed consumption / days; feed-to-meat ratio = feed consumption / weight gain.

[0196] The results showed (see Table 8): CC28×5g treatment increased ADG and ADFI of broilers in the early stage; increased ADFI of broilers throughout the entire period, significantly reduced F / G, and increased the survival rate of chickens.

[0197] Table 8 Effects of antimicrobial peptide CC28-cellulase powder on growth performance of broilers

[0198]

[0199]

[0200] Example 11: Determination of Apparent Metabolic Rate of Nutrients

[0201] Experimental animals and group treatment

[0202] This study used a single-factor, completely randomized design. Four hundred broilers in good condition and of similar body weight were randomly divided into four groups: a control group (CON), a CC28-cellulase 2.5g group (CC28×2.5g), a CC28-cellulase 5g group (CC28×5g), and a CC28-cellulase 7.5g group (CC28×7.5g). The CON group was fed a basal diet, while the CC28×2.5, CC28×5g, and CC28×7.5g groups were supplemented with 2.5, 5, and 7.5g / kg of the antimicrobial peptide CC28-cellulase additive powder, respectively, based on a target protein content of 41.59mg / g of engineered bacterial powder. Each group had six replicates, each containing 25 birds. Water was freely available and routine feeding and management were maintained for 42 days.

[0203] Nutrient metabolism tests were conducted on broilers at 3 and 6 weeks of age using the full fecal collection method. Dry matter (DM), crude protein (CP), crude fat (EE), neutral detergent fiber (NDF), acid detergent fiber (ADF), calcium (Ca), and phosphorus (P) in feed and fecal samples were determined according to the "Feed Analysis and Feed Quality Testing Technology" standard. Nutrient metabolism rates were calculated, and the results are shown in Table 9. The calculation formula is as follows:

[0204] Nutrient metabolism rate (%) = (C0 × T0 - C1 × T1) / C0 × T0. Where C0 represents the amount of feed consumed by broilers during the metabolic period; T0 represents the nutrient content in the feed; C1 represents the weight of dry manure; and T1 represents the nutrient content in the manure.

[0205] Table 9 Effect of antimicrobial peptide CC28-cellulase powder on nutrient utilization in broiler chickens (%)

[0206]

[0207] Results (Table 9) show that adding 5g of CC28 powder to the diet of Example 11 increased dry matter utilization by 3.34% in broiler chickens aged 22-42 days. Adding 2.5g and 5g of CC28 powder to the diet increased feed fat utilization by 3.52% and 4.0% in broiler chickens aged 1-21 days. At 22-42 days, the 5g powder showed a significant effect, increasing feed fat utilization by 7.2%.

[0208] Adding antimicrobial peptide additives increased the utilization rate of feed crude ash by 16.11%-23.56%. Adding 5g-7g of antimicrobial peptide additives to the 22-42 day old stage increased the utilization rate of feed protein by 7.05%-8.24%.

[0209] In the 1-21-day-old age group, a 7.5g dose of the antimicrobial peptide supplement increased the neutral detergent fiber utilization rate (eNDF) of chicken feed by 15.63%, while 7.5g and 5g increased the acid detergent fiber utilization rate (eADF) by 16.47% and 15.22% respectively. In the 22-42-day-old age group, a 5g dose of the antimicrobial peptide CC28 supplement increased the eNDF of broiler feed by 15.21%, while 7.5g increased the eADF of feed by 19.77%.

[0210] In the 1-21 day age stage, 2.5g and 5g of antimicrobial peptide additives increased the phosphorus utilization rate of chicken feed by 5.85% and 15.12%. In conclusion, the antimicrobial peptide CC28-cellulase powder had a better effect when added at a level of 5g / kg.

[0211] Example 12: Determination of slaughter performance and organ index

[0212] At 21 and 42 days of age, three birds were randomly selected from each group and replicate in the early morning on an empty stomach. Blood was collected from the jugular vein and then exsanguinated. Carcass performance was determined according to the methods for slaughter rate, evisceration rate, evisceration rate, breast muscle percentage, leg muscle percentage, and abdominal fat percentage as described in the "Terminology and Statistical Methods for Measurement of Poultry Performance" (NY / T823-2004). The heart, gizzard, proventriculus, pancreas, liver, thymus, spleen, and bursa of Fabricius were removed and weighed. Organ index = organ weight (g) / live weight (kg) before slaughter. The results are shown in Table 10.

[0213] (1) Carcass rate = (carcass weight + live weight) × 100%;

[0214] (2) Eviscerated rate = (Eviscerated weight + live weight) × 100%;

[0215] (3) Eviscerated rate = (eviscerated weight + live weight) × 100%;

[0216] The results showed (Table 10) that CC28 treatment increased the early slaughter rate of broilers; CC28×5g treatment increased the semi-eviscerated rate of broilers at 42 days and reduced the abdominal fat rate.

[0217] Table 10 Effects of antimicrobial peptide CC28-cellulase powder on the slaughter performance of broiler chickens

[0218]

[0219] Example 13: Determination of intestinal morphology and fecal colony count

[0220] At 21 and 42 days of age, three experimental chickens were randomly selected from each group and each replicate in the early morning on an empty stomach. The number of Escherichia coli and Staphylococcus aureus colonies in the feces of broiler chickens of different ages was determined. The results are shown in Table 11.

[0221] The results showed (Table 11) that CC28 treatment inhibited the number of Escherichia coli and Staphylococcus aureus colonies in broiler feces at different growth stages; and promoted the proliferation of Bifidobacterium.

[0222] Table 11 Effect of antimicrobial peptide CC28-cellulase powder on fecal bacterial counts in white-feathered broiler chickens (lg CFU / g)

[0223]

[0224] These experimental results show that the antimicrobial peptide CC28-cellulase engineered bacterial powder can improve the growth performance, apparent metabolic rate of nutrients, slaughter performance of broiler chickens, improve intestinal morphology and promote the proliferation of beneficial bacteria.

[0225] The above description is only a preferred specific embodiment of the present invention, and the protection scope of the present invention is not limited thereto. Any simple change or equivalent replacement of the technical solution that can be obviously obtained by any technician familiar with this technical field within the technical scope disclosed in the present invention falls within the protection scope of the present invention.

Claims

1. A novel tandem antimicrobial peptide CC28, characterized in that: The amino acid sequence of the novel tandem antimicrobial peptide CC28 is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.

3.

2. A CC28-cellulase protein, characterized in that The CC28-cellulase protein is assembled from the antimicrobial peptide CC28 and the ligand cellulase. The amino acid sequence of the CC28-cellulase protein is shown in SEQ ID NO.2, and the nucleotide sequence of the CC28-cellulase protein is shown in SEQ ID NO.6, which includes the antimicrobial peptide CC28 nucleotide sequence, the ligand sequence, and the linker sequence.

3. A recombinant Pichia pastoris engineered bacterium that secretes the CC28-cellulase protein according to claim 2, characterized in that: The preparation method of the recombinant Pichia pastoris engineered bacteria is as follows: subcloning the target protein gene into the pGAPZαA expression vector to construct a recombinant plasmid pGAPZαA / CC28-CE, and then transforming it into Pichia pastoris to obtain the recombinant Pichia pastoris engineered bacteria; the target protein gene is shown in SEQ ID NO.

6.

4. The recombinant plasmid pGAPZαA / CC28-CE constructed during the preparation of the recombinant Pichia pastoris engineered bacteria according to claim 3.

5. Use of the novel tandem antimicrobial peptide CC28 according to claim 1, or the CC28-cellulase protein according to claim 2, or the recombinant Pichia pastoris engineered bacteria according to claim 3, or the recombinant plasmid pGAPZαA / CC28-CE according to claim 4 in the preparation of an antimicrobial product, characterized in that: The bacteria targeted by the antibacterial product include Gram-negative bacteria and / or Gram-positive bacteria.

6. The use according to claim 5, characterized in that The Gram-negative bacteria include one or more of Escherichia coli, Salmonella enterica and Enterobacter aerogenes; the Gram-positive bacteria include Streptococcus agalactiae and / or Staphylococcus aureus.

7. The use according to claim 5, characterized in that The MIC value of the novel tandem antimicrobial peptide CC28 in the antimicrobial product against the Gram-negative bacteria and the Gram-positive bacteria is 1.11 μmol / L to 71.1 μmol / L.

8. Use of the novel tandem antimicrobial peptide CC28 according to claim 1, the CC28-cellulase protein according to claim 2, the recombinant Pichia pastoris engineered bacteria according to claim 3, or the recombinant plasmid pGAPZαA / CC28-CE according to claim 4 in the preparation of livestock feed additives.

9. Use of the novel tandem antimicrobial peptide CC28 according to claim 1, or the CC28-cellulase protein according to claim 2, or the recombinant Pichia pastoris engineered bacteria according to claim 3, or the recombinant plasmid pGAPZαA / CC28-CE according to claim 4 in the preparation of anticancer drugs.

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